The overall goal of this proposal is to unravel common cellular and molecular events underlying the altered excitability of neurons in mesial temporal lobe epilepsy (MTLE). The overall premise is that the more fundamental a change in neuronal excitability leading to MTLE, the more conserved across species and experimental models should be the underlying mechanisms. The present study will examine three essential molecular/cellular mechanisms regulating hippocampal neuronal excitability in chronic MTLE: 1) the role of the phosphoprotein phosphatase calcineurin (CaN) and the negative feedback it exerts on MDA channel openings; 2) the control of inhibitory activity by kainic acid (KA) receptors; and 3) the gating function of the dentate gyrus and its potential breakdown during the course of MTLE development. The experiments will be carried out in neurons and slices obtained from MTLE patients after surgery and form the kindling model of experimental MTLE. In addition, collaborations with other investigators, the possible involvement of the three mechanisms mentioned above will be systematically tested in the intrahippocampal KA injection model of MTLE. The first hypotheses centers on the role of CaN in MTLE. The related specific aims including examining changes in CaN function using physiological methods by determining the negative feedback exerted by CaN on the duration of NMDA receptor openings monitored in cell- attached or excised patch-clamp recordings in acutely isolated neurons. Biochemical measurements in brain slices will establish possible alterations in the activity and/or levels of CaN, including the role of a specific CaN inhibitory protein cain. Moreover, the epileptic phenotype of mice lacking the alpha isoform of the A subunit of CaN, will serve to further corroborate the role of CaN in the pathogenesis of MTLE. The second hypothesis concerns the possible contribution to MTLE mediated excitation of interneurons will be measured in models of MTLE using whole-cell recordings in brain slices. The third hypothesis relates to the gradual erosion of the normal dampening function of the dentate gyrus will be used to address experimentally this hypothesis in models of MTLE. In the intrahippocampal KA injection model of MTLE, it will be possible to study the dynamics of this erosion at distinct stages during the progression of MTLE. Understanding the fundamental mechanisms how neurons can sustain epileptic discharges, how they remain in this state, and how a lasting change in their excitability perturbs the epileptogenic zone will open new approaches aimed at restoring normal excitability in epileptogenic structures.